Paula C. Fernandez

MYC recruits the TIP60 histone acetyltransferase complex to chromatin

The transcription factor MYC binds specific DNA sites in cellular chromatin and induces the acetylation of histones H3 and H4. However, the histone acetyltransferases (HATs) that are responsible for these modifications have not yet been identified. MYC associates with TRRAP, a subunit of distinct macromolecular complexes that contain the HATs GCN5/PCAF or TIP60. Although the association of MYC with GCN5 has been shown, its interaction with TIP60 has never been analysed. Here, we show that MYC associates with TIP60 and recruits it to chromatin in vivo with four other components of the TIP60 complex: TRRAP, p400, TIP48 and TIP49. Overexpression of enzymatically inactive TIP60 delays the MYC‐induced acetylation of histone H4, and also reduces the level of MYC binding to chromatin. Thus, the TIP60 HAT complex is recruited to MYC‐target genes and, probably with other other HATs, contributes to histone acetylation in response to mitogenic signals.

Theileria parva: taking control of host cell proliferation and survival mechanisms.

The intracellular parasite Theileria parva infects and transforms bovine T‐cells, inducing their uncontrolled proliferation and spread in non‐lymphoid as well as lymphoid tissues. This parasite‐induced transformation is the predominant factor contributing to the pathogenesis of a lymphoproliferative disease, called East Coast fever. T. parva‐transformed cells become independent of antigenic stimulation or exogenous growth factors. A dissection of the signalling pathways that are activated in T. parva‐infected cells shows that the parasite bypasses signalling pathways that normally emanate from the T‐cell antigen receptor to induce continuous proliferation. This review concentrates on the influence of the parasite on the state of activation of the mitogen‐activated protein kinase (MAPK), NF‐κB and phosphoinositide‐3‐kinase (PI3‐K) pathways in the host cell. Of the MAPKs, JNK, but not ERK or p38, is active, inducing constitutive activation of the transcription factors AP‐1 and ATF‐2. A crucial step in the transformation process is the persistent activation of the transcription factor NF‐κB, which protects T. parva‐transformed cells from spontaneous apoptosis accompanying the transformation process. Inhibitor studies also suggest an important role for the lipid kinase, PI‐3K, in the continuous proliferation of T. parva‐transformed lymphocytes.

N-acetylcysteine blocks apoptosis induced by N-alpha-tosyl-L-phenylalanine chloromethyl ketone in transformed T-cells

The serine protease inhibitor N-α-tosyl-L-phenylalanine chloromethyl ketone (TPCK) can interfere with cell-cycle progression and has also been shown either to protect cells from apoptosis or to induce apoptosis. We tested the effect of TPCK on two transformed T-cell lines. Both Jurkat T-cells and Theileria parva-transformed T-cells were shown to be highly sensitive to TPCK-induced growth arrest and apoptosis. Surprisingly, we found that the thiol antioxidant, N-acetylcysteine (NAC), as well as L- or D-cysteine blocked TPCK-induced growth arrest and apoptosis. TPCK inhibited constitutive NF-κB activation in T. parva-transformed T-cells, with phosphorylation of IκBα and IκBβ being inhibited with different kinetics. TPCK-mediated inhibition of IκB phosphorylation, NF-κB DNA binding and transcriptional activity were also prevented by NAC or cysteine. Our observations indicate that apoptosis and NF-κB inhibition induced by TPCK result from modifications of sulphydryl groups on proteins involved in regulating cell survival and the NF-κB activation pathway(s).

The inhibition of NF-kappaB activation pathways and the induction of apoptosis by dithiocarbamates in T cells are blocked by the glutathione precursor N-acetyl-L-cysteine

Abstract Nuclear factor-κB regulates genes that control immune and inflammatory responses and are involved in the pathogenesis of several diseases, including AIDS and cancer. It has been proposed that reactive oxygen intermediates participate in NF-κB activation pathways, and compounds with putative antioxidant activity such as N-acetyl-L-cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC) have been used interchangeably to demonstrate this point. We examined their effects, separately and combined, on different stages of the NF-κB activation pathway, in primary and in transformed T cells. We show that NAC, contrary to its reported role as an NF-κB inhibitor, can actually enhance rather than inhibit IκB degradation and, most importantly, show that in all cases NAC exerts a dominant antagonistic effect on PDTC-mediated NF-κB inhibition. This was observed at the level of IκB degradation, NF-κB DNA binding, and HIV-LTR-driven reporter gene expression. NAC also counteracted growth arrest and apoptosis induced by dithiocarbamates. Antagonistic effects were further observed at the level of jun-NH2-terminal kinase, p38 and ATF-2 activation. Our findings argue against the widely accepted assumption that NAC inhibits all NF-κB activation pathways and shows that two compounds, previously thought to function through a common inhibitory mechanism, can also have antagonistic effects.

Characterisation of NF-κB complexes in Theileria parva-transformed T cells

Abstract Transformation of T cells by the intracellular parasite Theileria parva is accompanied by constitutive I-κB degradation and NF-κB activation, a process which is essential to prevent the spontaneous apoptosis of these parasite-transformed cells. NF-κB-mediated responses are regulated by selective combinations of NF-κB proteins as homo- or heterodimers and by distinct κB motifs. We characterised the NF-κB complexes induced by T. parva infection in TpM(803) T cells. By western blot, we demonstrated that all members of the NF-κB/Rel family of proteins translocate to the nucleus of infected cells. Using two different κB oligonucleotides (κB-1 and κB-2), both containing the decameric consensus κB motif (GGGACTTTCC), clearly distinct patterns of DNA binding activities could be demonstrated in electrophoretic mobility shift assays. Supershift analysis and UV cross-linking assays showed that complexes binding to κB-1 consisted of p50, p65 and RelB homo and/or heterodimers. We could also detect an association of ATF-2 and c-Fos with one of the complexes. The HIV-derived κB-2 oligo only bound p50 and p65. Additionally, several agents known to inhibit a wide range of NF-κB activation pathways had no inhibitory effect on the activation of NF-κB DNA binding in TpM(803) T cells.

Interference by the intracellular parasite Theileria parva with T-cell signal transduction pathways induces transformation and protection against apoptosis.

The intracellular parasite Theileria parva transforms bovine T-lymphocytes, inducing uncontrolled proliferation. Upon infection, cells cease to require antigenic stimulation and exogenous growth factors to proliferate. Earlier studies have shown that pathways triggered via stimulation of the T-cell receptor are silent in transformed cells. This is reflected by a lack of phosphorylation of key signalling molecules and the fact that proliferation is not inhibited by immunosuppressants such as cyclosporin and ascomycin that target calcineurin. This suggests that the parasite bypasses the normal T-cells activation pathways to induce proliferation. Among the MAP-kinase pathways, ERK and p38 are silent, and only Jun N-terminal kinase is activated. This appears to suffice to induce constitutive activation of the transcription factor AP-1. More recently, it could be shown that the presence of the parasite in the host cell cytoplasm also induces constitutive activation of NF-kappaB, a transcription factor involved in proliferation and protection against apoptosis. Activation is effectuated by parasite-induced degradation of IkappaBs, the cytoplasmic inhibitors which sequester NF-kappaB in the cytoplasm. NF-kappaB activation is resistant to the antioxidant N-acetyl cysteine and a range of other reagents, suggesting that activation might occur in an unorthodox manner. Studies using inhibitors and dominant negative mutants demonstrate that the parasite activates a NF-kappaB-dependent anti-apoptotic mechanism that protects the transformed cell form spontaneous apoptosis and is essential for maintaining the transformed state of the parasitised cell.

The IKK Inhibitor BMS-345541 Affects Multiple Mitotic Cell Cycle Transitions

The IκB kinase (IKK) complex controls processes such as inflammation, immune responses, cell survival and the proliferation of both normal and tumor cells. By activating NFκB, the IKK complex contributes to G1/S transition and first evidence has been presented that IKKα also regulates entry into mitosis. At what stage IKK is required and whether IKK also contributes to progression through mitosis and cytokinesis, however, has not yet been determined. In this study, we use BMS-345541, a potent allosteric small molecule inhibitor of IKK, to inhibit IKK specifically during G2 and during mitosis. We show that BMS-345541 affects several mitotic cell-cycle transitions, including mitotic entry, prometaphase to anaphase progression and cytokinesis. Adding BMS-345541 to the cells released from arrest in S-phase blocked the activation of aurora A, B and C, Cdk1 activation and histone H3 phosphorylation. Additionally, treatment of the mitotic cells with BMS-345541 resulted in precocious cyclin B1 and securin degradation, defective chromosome separation and improper cytokinesis. BMS-345541 was also found to override the spindle checkpoint in nocodazole-arrested cells. In vitro kinase assays using BMS-345541 indicate that these effects are not primarily due to a direct inhibitory effect of BMS-345541 on mitotic kinases such as Cdk1, Aurora A or B, Plk1 or NEK2. This study points towards a new potential role of IKK in cell cycle progression. Since deregulation of the cell-cycle is one of the hallmarks of tumor formation and progression, the newly discovered level of BMS 345541 function could be useful for cell-cycle control studies and may provide valuable clues for the design of future therapeutics.